Measuring Biological Responses with Automated Microscopy 1st Edition by James Inglese ISBN 0121828190 9780121828196

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ISBN 10: 0121828190 
ISBN 13: 9780121828196
Author: James Inglese 

The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today-truly an essential publication for researchers in all fields of life sciences.

Measuring Biological Responses with Automated Microscopy 1st Table of contents:

Chapter 1: Dynamic Green Fluorescent Protein Sensors for High- Content Analysis of the Cell Cycle
Introduction
Design and Construction of Dynamic Cell Cycle Sensors
Validation of Sensors
Cell Cycle Analysis by Automated High-Throughput Imaging for Drug Profiling and Target Validation
Conclusions and Future Directions
Acknowledgments
References
Chapter 2: High-Content Fluorescence-Based Screening for Epigenetic Modulators
Introduction
Epigenetic Regulators of Gene Expression as Drug Targets
Rationale for the Development of Cell-Based Assays to Screen for Epigenetic Modulators
Methodological Considerations
General Conclusions and Perspectives
References
Chapter 3: Development of Assays for Nuclear Receptor Modulators Using Fluorescently Tagged Proteins
Introduction
Methods
Perspectives and Future Applications
References
Chapter 4: The Ligand-Independent Translocation Assay: An Enabling Technology for Screening Orphan G
Introduction
Overview of the LITe Assay
Utility of the LITe Assay
Conclusion
Acknowledgments
References
Chapter 5: High-Content Screening of Known G Protein-Coupled Receptors by Arrestin Translocation
Introduction
Stable Expression of a Known GPCR for the ArrestinGFP Translocation Assay
Screening Known Receptors with Transfluor Technology
Conclusion
Acknowledgments
References
Chapter 6: Cell Imaging Assays for G Protein-Coupled Receptor Internalization: Application to High-T
Introduction
Monitoring GPCR Trafficking via Receptor-GFP
Monitoring GPCR Trafficking via Receptor-Arrestin-GFP
Case I: MRG-X1 Receptor
Case Study 2: NK1 Receptor Trafficking
Acknowledgments
References
Chapter 7: High-Throughput Confocal Microscopy for ß-Arrestin-Green Fluorescent Protein Translocati
Choice of Orphans
Transfluor
Technology
Image Analysis Algorithm
EC50 Fitting and Scoring
Assay Methods
Screen Metrics and Results
Concluding Remarks
References
Chapter 8: G Protein-Coupled Receptor Internalization Assays in the High-Content Screening Format
Introduction
High-Throughput Confocal Cellular Imaging Systems
GPCR Internalization Assays
Internalization Assay and Image Analysis Protocols
Conclusions and Outlook
Acknowledgment
References
Chapter 9: Screening for Activators of the Wingless Type/Frizzled Pathway by Automated Fluorescent M
Introduction
Use of Primary Human Preosteoblasts
Reagents and Materials
Preparation of L Cell Control- and Wnt3A-Conditioned Medium
Primary Preosteoblast Cell Culture and Compound Screening
Immunofluorescent Staining
Imaging and Quantitation of Nuclear Translocation
Data Analysis
Acknowledgments
References
Chapter 10: A Live Cell, Image-Based Approach to Understanding the Enzymology and Pharmacology of 2-
Introduction
Types of Protein Lipid Modifications
Palmitate Turnover
Enzymes for Depalmitoylation
Role of Palmitoylation in Development
Cysteines: Primary Sites of Palmitoylation
Palmitoyl Acyl Transferases (PATs)
Links between Palmitoylation and Disease
Using Fluorescent Proteins to Study Protein Lipid Modifications
Monomeric Fluorescent Proteins: A Critical Feature for Studying Palmitoylation
High-Throughput Microscopy (HTM)/High-Content Screening (HCS) to Study Lipid-Modified Proteins
Morphometric Analysis of Palmitoylation with HCS
HCS Machine Vision Algorithms to Quantify Reporter Density on the Plasma Membrane
GAP43:YFP is Palmitoylated, Localized to the Plasma Membrane, and Is a Stereotypical Reporter of the
Determination of the Residence Half-Life of Palmitate on GAP43:YFP Using HCS
Thora Can Measure Precisely the Subcellular Distribution of GAP43:YFP: IC50 of 2BP
Determination of the Compatibility of the Cellular Reporter System with Dimethyl Sulfoxide (DMSO)
Cytotoxic Effects of Antagonists of Palmitoylation
Considerations for Designing HCS, Cell-Based Assays with an Emphasis on Palmitoylation
Troubleshooting
System Error Identification
Data Tracking
Image Organization and Analysis
Conclusions
References
Chapter 11: High-Resolution, High-Throughput Microscopy Analyses of Nuclear Receptor and Coregulator
High-Throughput Microscopy (HTM)
Steroid Nuclear Receptors and Coregulators
General Methods
Nuclear Receptor Coregulator SRC-3
Protein Expression Level Assay
Protein Translocation and Nuclear Variance Assays
Foci Identification and Chromatin Remodeling Assay
Conclusions
References
Chapter 12: Tracking Individual Proteins in Living Cells Using Single Quantum Dot Imaging
Introduction
Semiconductor Quantum Dots as Biological Probes
Single Molecule Imaging in Living Cells
Single Quantum Dot Tracking of Membrane Proteins
Solutions and Materials
Methods
Single Quantum Dot Tracking of Intracellular Proteins
Solutions and Materials
Methods
Imaging of Single Conjugated Quantum Dot in Living Cells
Analysis of Single QD trajectories
Image Processing and Extraction of Single QD Trajectories
Mean Square Displacement Function
Statistical Errors
Acknowledgments
References
Chapter 13: Development and Application of Automatic High-Resolution Light Microscopy for Cell-Based
Introduction
Technical Description
Prospectives
Acknowledgments
References
Chapter 14: Adenoviral Sensors for High-Content Cellular Analysis
Introduction
Design and Construction of Adenoviral Sensors for Cellular Assays
Validation of Adenoviral Sensors
Application of Adenoviral Sensors to High-Content Analysis
EGFP-Glucocorticoid Receptor (GR) Translocation Sensor
Reagent Preparation
Assay Procedure
Assay Analysis
Nuclear Factor of Activated T Cells (NFAT) Nitroreductase Reporter Gene Sensor
Reagent Preparation
Assay Procedure
Assay Analysis
Conclusions and Future Perspectives
Acknowledgments
References
Chapter 15: Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammatio
Cellular Mechanism of the Inflammatory Response
Cell-Based Assays Used to Monitor the Effects of Proinflammatory Cytokines
Materials and Methods Common to All Three Assays
Image Analysis
Statistical Analysis
Assay Data
Concluding Remarks and Future Perspectives
Acknowledgments
References
Chapter 16: Development and Implementation of Multiplexed Cell-Based Imaging Assays
Introduction
General Considerations
Monitoring Cell Cycle Progression
Protocols for Cell Cycle Analysis
Visualizing Apoptosis by Fluorescence Microscopy
Multiplexing Cell Cycle and Apoptosis Assays
Conclusions
Acknowledgments
References
Chapter 17: High-Throughput Screening for Modulators of Stem Cell Differentiation
Introduction
Assay Design
Assay Development
Methods
Conclusion
Appendix
Acknowledgments
References
Chapter 18: High-Content Kinetic Calcium Imaging in Drug-Sensitive and Drug-Resistant Human Breast C
Introduction
High-Content Analysis of Ca2+ Dynamics by Fluorescence Microscopy
Conclusions from HCA and Rationale for High-Content Screening
High-Content Screening
Acknowledgments
References
Chapter 19: Measurement and Analysis of Calcium Signaling in Heterogeneous Cell Cultures
Introduction
Characterization of Calcium Signaling in Rat Cortical Cultures
Subpopulation Analysis of Calcium Signaling in Cocultures
Acknowledgments
References
Chapter 20: Multiplex Analysis of Inflammatory Signaling Pathways Using a High-Content Imaging Syste
Introduction
Assay Procedures
Concluding Remarks
Acknowledgments
References
Chapter 21: Generation and Characterization of a Stable MK2- EGFP Cell Line and Subsequent Developme
Introduction
Definition of the Cell Model
Generation and Characterization of MK2-EGFP HeLa Cell Line
Cellomics ArrayScan Automated Imaging Platform
Characterization of MK2-EGFP Clones via Imaging
Characterization of the p38 MAPK Signaling Pathway in HeLa-MK2-EGFP Cells
Assay Development on the ArrayScan 3.1 Imaging Platform
Standard Operating Procedure for the MK2-EGFP Translocation Assay
MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation
p38 Inhibitor Data
Secondary Analysis Parameters
Discussion
References
Chapter 22: Development and Implementation of Three Mitogen-Activated Protein Kinase (MAPK) Signalin
Introduction
Definition of the Cell Model
Cellomics ArrayScan Automated Imaging Platform
Development of the JNK MAPK Signaling Pathway Assay
c-Jun Activation Protocol
c-Jun Activation Signal Window and Reproducibility
Development of the ERK MAPK Signaling Pathway Assay
ERK1/2 Activation Protocol
ERK1/2 Activation Signal Window and Reproducibility
MAPK Pathway Inhibitor Test Cassette
p38 Inhibitor Hit Assessment
p38a Inhibitor Profiling
JNK Inhibitor Profiling
Discussion
References
Chapter 23: Assay Development and Case History of a 32K-Biased Library High-Content MK2-EGFP Translo
Introduction
Cellomics ArrayScan Automated Imaging Platform
Conversion of the 96-Well MK2-EGFP Translocation Assay to a 384-Well Format Assay on the Arrayscanre
MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation
Standardized Operation Procedure for the MK2-EGFP Translocation Assay
MK2-EGFP Translocation HTS Assay for p38 Inhibitors
Discussion
References
Chapter 24: Compound Classification Using Image-Based Cellular Phenotypes
Introduction
Quantifying Cellular Morphology Changes
Cell Culture, Compound Addition, and Image Acquisition
Image and Data Reduction
Analysis of Quantitative Cellular Phenotypes across Cell Lines
Clustering and Classification of Compounds
Conclusions
Acknowledgments
References
Chapter 25: High-Content Screening: Emerging Hardware and Software Technologies
Introduction
Cellular Assay and Imaging Preparation
Image Acquisition
Image Analysis
Image Database and Data Visualization Tools
Conclusion
Protocols
References
Chapter 26: An Infrastructure for High-Throughput Microscopy: Instrumentation, Informatics, and Inte
Introduction
Assay Processing
Image Acquisition
Image and Data Analysis
Data Review and Quality Control
Summary
Acknowledgments
References
Chapter 27: Protein Translocation Assays: Key Tools for Accessing New Biological Information with Hi
Pathway Screening Using BioImage Redistribution Technology
p53-Hdm2 Protein-Protein Interaction Assay
Use of High-Content Assays in RNAi Studies
Use of siRNA-Mediated Knockdown to Validate Akt Isoform Dependency of a FKHR Redistribution Assay
Assay-Specific Cell-to-Cell Heterogeneity Plays a Role in Assay Quality
Future Developments
References
Chapter 28: High-Content Screening of Functional Genomic Libraries
Introduction
Development of Large-Scale Genomic Libraries
Maintaining Large Arrayed-Well Plasmid cDNA or shRNA Clone Libraries
Collection Replication
Growth of Bacterial Cultures in High-Throughput Format for DNA Preparation
Preparing DNA from 96-Well Deep-Well Block Cultures
Normalization of Plasmid DNA
Arraying Collections into High-Throughput Assay Plates
High-Throughput Transfections
High-Throughput Retroviral/Lentivial Packaging
Instrumentation Required for Functional Genomics Screening
Automated Microscopy
High-Throughput HCS Equipment
Fluorescent Biomarkers for HCS Applications
Preparing Samples for Automated Microscopy
Determining Transfection or Transduction Efficiency
Quantitative Image Analysis
Summary
References
Chapter 29: Fluorescent Protein-Based Cellular Assays Analyzed by Laser-Scanning Microplate Cytometr
Introduction
The Principle of Laser-Scanning Microplate Cytometers
General Methods
GR-GFP Nuclear Translocation Assay
Locus Derepression Assay
ß-Arrestin:ß2-Adrenergic Receptor (ßARR:ß2AR) Protein Fragment Complementation Assay
Summary
Acknowledgments
References
Chapter 30: High-Throughput Measurements of Biochemical Responses Using the Plate::Vision Multimode
Introduction
Instrumentation
Measurements of Biochemical Responses
Acknowledgments
References
Chapter 31: Systems Cell Biology Based on High-Content Screening
Background
The Systems Cell Biology Toolbox
Example Systems Cell Biology Profile
Summary and Conclusions
Prospectus
Acknowledgments
References
Chapter 32: Digital Autofocus Methods for Automated Microscopy
Introduction
Hardware
Software
Viscoelasticity
Multiple-Field Scans
Conclusion
Appendix
References
Chapter 33: Fluorescence Lifetime Imaging Microscopy: Two-Dimensional Distribution Measurement of Fl
Introduction
Operating Principle of a Streak Camera
Configuration of the FLIM System
Streak Image
Measurement Principle of FLIM System
System Calibration
Lifetime Imaging in Cells
FRET Imaging in Cells

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